Introduction: The number of patients with chronic kidney disease (CKD) is increasing worldwide. Cognitive impairment is one of the comorbidities of CKD. With the increased number of aged population, novel biomarkers of impaired cognitive function are required. Intra-body profile of amino acid (AA) is reportedly altered in patients with CKD. Although some AAs act as neurotransmitters in the brain, it is not clear whether altered AA profile are associated with cognitive function in patients with CKD. Therefore, intra-brain and plasma levels of AAs are evaluated with respect to cognitive function in patients with CKD. Methods: Plasma levels of AAs were compared between 14 patients with CKD, including 8 patients with diabetic kidney disease, and 12 healthy controls to identify the alteration of specific AAs in CKD. Then, these AAs were evaluated in the brains of 42 patients with brain tumor using non-tumor lesion of the resected brain. Cognitive function is analyzed with respect to intra-brain levels of AAs and kidney function. Moreover, plasma AAs were analyzed in 32 hemodialyzed patients with/without dementia. Results: In patients with CKD, plasma levels of asparagine (Asn), serine (Ser), alanine (Ala), and proline (Pro) were increased as compared to patients without CKD. Among these AAs, L-Ser, L-Ala, and D-Ser show higher levels than the other AAs in the brain. Intra-brain levels of L-Ser was correlated with cognitive function and kidney function. The number of D-amino acid oxidase or serine racemase-positive cells was not correlated with kidney function. Moreover, the plasma levels of L-Ser are also decreased in patients with declined cognitive function who are treated with chronic hemodialysis. Conclusion: The decreased levels of L-Ser are associated with impaired cognitive function in CKD patients. Especially, plasma L-Ser levels may have a potential for novel biomarker of impaired cognitive function in patients with hemodialysis.

© 2023 The Author(s). Published by S. Karger AG, Basel

Introduction

Chronic kidney disease (CKD) is a major contributor to mortality and morbidity worldwide. The estimated global all-age mortality rate from CKD increased by 41.5% between 1990 and 2017. Moreover, disability-adjusted life years for CKD were estimated at 35.8 million in 2017 [1]. Some comorbidities, such as hypertension, anemia, electrolyte disorder, and heart failure, are accompanied by CKD. Also, cognitive impairment and dementia are significant comorbidities. The prevalence of these diseases is 2–7 times higher in CKD patients as compared to the general population [2-4]. Therefore, to understand the mechanisms and develop novel biomarkers are highly required.

We have reported that ischemia-reperfusion kidney injury induces dysbiosis of the gut microbiota. Moreover, gut microbiota derived D-Serine (Ser) has a reno-protective effect on the ischemia-reperfusion-induced kidney injury [5]. CKD is also related to the alteration of microbiota component in gut. Major source of uremic toxins, such as indoxyl sulfate and p-cresyl sulfate, is reported to be the gut microbiota in CKD patients. The increased levels of uremic toxins have a potential to induce multiple organ damage including cardiovascular diseases [6-8]. These findings indicate that gut microbiota-derived metabolites are involved in the regulation of organ function in patients with kidney dysfunction.

AA imbalance has been reported in patients with end-stage kidney diseases (ESKD) [9, 10]. The impaired metabolism of dietary protein leads to the altered AA flow into the body in ESKD [11]. Also, some kinds of the patient meals for kidney disease show imbalanced AA’s pattern [12]. In fact, rats fed with low protein diet exhibited imbalanced AA in blood and brain [13]. Because some AAs, such as L-glutamate, L-aspartate, L-glycine, and d-/L-Ser, act as neurotransmitter, the impairment of AA balance reportedly causes neurological disorders [14]. However, it is not clear whether the alteration of AA’s composition is associated with mental status in CKD patients. Moreover, it also remains to be investigated whether AAs in circulation may reflect intra-brain AAs in CKD patients. Therefore, we evaluated the correlation of intra-body levels of AAs with cognitive status and explored the potential of plasma AAs as a novel biomarker for cognitive status in CKD patient.

Subjects and MethodsPatients

Fourteen patients with CKD, including 8 patients with diabetic kidney disease (DKD), and 12 healthy controls were enrolled for analyzing plasma and urine levels of AAs. The patients were visited or admitted to the Department of Nephrology and Laboratory Medicine in Kanazawa University Hospital. For intra-brain AAs’ analysis, we retrospectively enrolled 42 Japanese subjects who underwent brain tumor resection and were diagnosed with glioma, one of the most common types of primary brain tumors. The patients were admitted to the Department of Neurosurgery, Kanazawa University Hospital until 2018. We analyzed the levels of AAs using normal brain tissue around the tumor. All the patients’ characteristics are described in Table 1. Thirty-two patients with hemodialysis were enrolled in this study. Among them, the causes of ESKD were DKD in 16 patients and non-DKD in 16 patients, respectively. Hemodialysis was performed at Mizuho Hospital, and the samples were collected in 2019. All the HD patients’ characteristics are described in Table 2.

Table 1.

Characteristics of patients with brain resection

Table 2.

Characteristics of patients

Determination of Chiral Amino Acid by 2D HPLC

The concentrations of D- and L-amino acids were evaluated as previously reported (5). Briefly, the NBD-amino acids were isolated using a KSAARP column (1.0 mm i.d. × 500 mm, an ODS column designed by collaboration with Kyushu University and Shiseido) and an online fraction collecting system in the first dimension. The isolated fractions were automatically transferred to the second dimension, which consisted of a narrow-bore enantio-selective KSAACSP-001S column (250 mm × 1.5 mm i.d., 25 C; prepared in collaboration with Shiseido), to determine the D- and L-enantiomers. The mobile phases for the second dimension were mixed solutions of MeOH and MeCN that contained formic acid. The fluorescence detection of the NBD-amino acids was performed at 530 nm with an excitation at 470 nm.

Evaluation Assessment of Cognitive Function

We used two kinds of assessments to evaluate cognitive function, including the Japanese Adult Reading Test (JART) and Hasegawa’s dementia scale (HDS-R). The JART is an assessment for estimating premorbid IQ based on the oral reading of 25 kanji characters, which are Japanese characters. The test has been validated with good reliability and adequacy and is highly correlated with the Wechsler Adult Intelligence Scale, which is widely used worldwide assessment of intelligence [15]. The HDS-R which shows high correlation to the mini-mental state examination is the most fluently used as an assessment of cognitive function in Japan and can detect the presence of cognitive dysfunction with a sensitivity of 90% and a specificity of 82%, using a cutoff of 20/21 points [16].

Immunohistochemistry

We examined 8 patients’ normal brain tissue around the tumor, which was fixed in 4% paraformaldehyde and embedded in paraffin for routine histopathological and immunohistochemical analysis. The histological diagnosis was determined according to the revised World Health Organization criteria [17].

In brief, 4-μm-thick tissue sections were stained using the standard technique for hematoxylin and eosin staining. These slides were immunostained using the Envison+ System (Dako, Tokyo, Japan). After deparaffinization using Fast Solve and autoclaving at 120°C for 10 min in Target Retrieval Solution, pH 6.0 (Dako, Glostrup, Denmark), sections were quenched using 3% hydrogen peroxide (H2O2) in methanol for 20 min and blocked with 5% skim milk in TBST. Sections were incubated with 1:200 DAO rabbit polyclonal antibody (SIGMA, HPA038654) and 1:200 Serine racemase (A-4) mouse monoclonal antibody (Santa Cruz SC-365217), respectively, for overnight at 4°C. After washing with TBST, the corresponding secondary antibody was applied at room temperature for 1 h, and color was developed using 3,3-diaminobenzidine tetrahydrochloride (DAB Substrate Kit SK-4100, Vector, Japan) for 2–5 min. Sections were then counterstained with hematoxylin (Wako Pure Chemical Industries Ltd.). Images were acquired with a BZ-X700 microscope (Keyence, Osaka, Japan). For the respective molecules, the mean percentage of immunohistochemistry-positive normal cells in 3 microscopic fields was calculated for each normal brain sections.

Study Approval

This study was approved by the Ethics Committee of Kanazawa University Hospital and Mizuho Hospital (IRB approval no. 2893). The research was conducted in accordance with the Declaration of Helsinki. All participants provided written informed consent and were informed about their right to withdraw from the study at any time.

Statistics

The data were presented as mean ± standard error of the mean, as determined by the SPSS Statistics version 23 software (IBM, Tokyo). Statistical analysis was performed using a two-tailed unpaired Student’s t test when comparing two groups. A one-way ANOVA with the Tukey multiple comparison test was used when comparing multiple groups. In all legends and figures, significance was set at *p < 0.05, **p < 0.01, and ***p < 0.001.

ResultsThe Plasma Levels of Asparagine, Ser, Alanine, and Proline Were Increased in CKD Patients

We evaluated plasma and urine levels of AAs with 2D HPLC of the patients with/without CKD. In the patients with CKD, plasma levels of D-asparagine (Asn), -Ser, -alanine (Ala), and -proline (Pro) and L-Asn, -Ala, and -Pro were increased as compared to patients without CKD (Fig. 1). In contrast, urine levels of D-Asn and -Ser, and L-Asn and -Ser were decreased in patients with CKD, suggesting that renal excretion may contribute to the plasma levels of these AAs (online suppl. Fig. 1; for all online suppl. material, see www.karger.com/doi/10.1159/000527798).

Fig. 1.

The plasma levels of amino acids in CKD patients. Levels of D- (a) and L-amino acids (b) in plasma from CKD patients and healthy control were analyzed with 2D HPLC. c Also, the percentage of D-amino acid were shown. Data are shown as means ± SEM. Statistical analysis was performed using Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001.

L-/D-Amino Acids Were Distributed in Brain

Because the levels of D-/L-Ser, Ala, Asn, and Pro were increased in plasma, we evaluated the intra-brain levels of these AAs in the patients with CKD. Among these, L-Ser, L-Ala, and D-Ser show higher levels than the other AAs (Fig. 2a, b). Also, the percentage of D-Ser is higher than those of d-/L-Ala, D/L-Asn, and D/L-Pro (Fig. 2c).

Fig. 2.

The evaluation of intra-brain levels of D-/L-Ser, -Ala, -Asn, and -Pro. Intra-brain levels of D-Ser, -Ala, -Asn, and -Pro (a) and L-Ser, -Ala, -Asn, and -Pro (b) were evaluated. c Also the percentage of D-Ser, -Ala, -Asn, and -Pro are shown. Statistical analysis was performed using Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001.

Intra-Brain Levels of L-Ser Are Correlated with Kidney Function

Next, we evaluated the association between intra-brain levels of D-/L-amino acids and kidney function. The intra-brain levels of L-Ser were significantly correlated with an increase in creatinine and a decrease in estimated glomerular filtration rate (eGFR) (Fig. 3a). No correlation was detected between intra-brain levels of D-Ser and kidney function. Considering that plasma levels of L-Ser were similar between the patients with/without CKD, the difference in intra-brain levels of L-Ser may not be affected by the plasma levels of L-Ser (Fig. 1, 3). Moreover, plasma levels of L-Ser were not different between the CKD with/without diabetes (online suppl. Fig. 2), suggesting that diabetes itself does not affect the levels of L-/D-Ser in CKD patients. In addition, intra-brain D-Asn level was inversely correlated with kidney function (Fig. 3b).

Fig. 3.

The correlation between intra-brain levels of L-Ser and kidney function. a The association between intra-brain L-AAs levels and kidney functions were evaluated. Intra-brain levels of L-Ser were correlated with kidney function. b Also, the association of intra-brain D-AAs levels and kidney functions were evaluated. Intra-brain levels of D-Asn were correlated with kidney function.

JART Score Was Correlated with Intra-brain Levels of L-Ser and Kidney Function

To explore the relationship among cognitive function, kidney function, and intra-brain levels of L-Ser, JART was performed in these patients. Interestingly, the patients both with the eGFR of lesser than 90 mL/min/1.73 m2 and intra-brain L-Ser of lower than 600 nmol/g tended to show the lower JART score than those with the eGFR of higher than 90 mL/min/1.73 m2 or intra-brain L-Ser of more than 600 nmol/g (Fig. 4a). Also, JART score was associated with kidney function, indicated by serum creatinine and eGFR in patients with eGFR of less than 90 mL/min/1.73 m2 (Fig. 4b). Moreover, JART score is positively correlated with intra-brain levels of L-Ser (Fig. 4c). No correlation was detected between the JART score and intra-brain levels of D-Ser.

Fig. 4.

The correlation between levels of intra-brain L-Ser and JART score in patients with lower kidney function. a The patients both with the eGFR of lesser than 90 mL/min/1.73 m2 and intra-brain L-Ser of lower than 600 nmol/g tended to show the lower JART score than those with the eGFR of higher than 90 mL/min/1.73 m2 or intra-brain L-Ser of more than 600 nmol/g. b Also, JART score was associated with kidney function, indicated by serum creatinine and eGFR in patients with eGFR of lesser than 90 mL/min/1.73 m2. c JART score is positively correlates with intra-brain levels of L-Ser. No correlation was detected between JART score and intra-brain levels of D-Ser.

The Number of DAO or SRR Positive Cells Was Not Correlated with Renal Function

The metabolization of L-Ser and D-Ser were closely linked with each other. D-Ser is synthesized from L-Ser by SRR [18]. The increased levels of L-Ser also induce the increased levels of D-Ser in the rat brain [19]. In addition, D-Ser is metabolized by DAO [5]. These findings raised the possibility that the decreased concentration of L-Ser is due to the alteration of SRR and/or DAO expression levels in the brain of CKD patients. To explore this, we performed immunohistochemistry of SRR and DAO in brain. DAO and SRR are detected on astrocytes and neurons in the brain, respectively (Fig. 5a). The frequency of DAO positive cells is not correlated with kidney function. Also, the frequency of SRR-positive cells is not correlated with kidney function (Fig. 5b).

Fig. 5.

Intra-brain expressions of DAO and SRR. a DAO and SRR are detected on astrocyte and neuron in brain, respectively. Representative images of tissue samples stained with HE, DAO, and SRR are shown. Scale bar, 100 μm. Arrowheads indicate DAO positive astrocyte. Arrows indicate SRR positive neuron. b The frequency of DAO positive cells is not correlated with kidney function. Also, the frequency of SRR positive cells is not correlated with kidney function.

The Plasma Levels of L- and D-Ser Are Also Decreased in Patients with Declined Cognitive Function, Who Are Treated with Chronic Hemodialysis

To exclude the effect of kidney function for the concentration of L-/D-Ser, we compared the plasma levels of L- and D-Ser in patients who were treated with chronic hemodialysis. Moreover, diabetes is also the risk for cognitive decline. Therefore, we performed the analysis in hemodialyzed patients with DKD and those without DKD separately. The plasma levels of L-Ser were also lower in the hemodialyzed patients with cognitive decline as compared to those with non-cognitive impairment, both in patients with/without DKD (Fig. 6). Also, the percentage of D-Asn and D-Pro was higher in non-DKD patients with dementia as compared to those without dementia (Fig. 6).

Fig. 6.

The plasma levels of D-/L-Ser, Ala, Asn, and Pro in hemodialyzed patients with/without dementia. The comparison of plasma levels of D-/L-Ser, Ala, Asn, and Pro in hemodialyzed patients with/without dementia. The plasma levels of L-Ser were also lower in the hemodialyzed patients with cognitive decline as compared to those with non-cognitive impairment both in patients with/without DKD. AAs were analyzed with 2D HPLC. A score of less than 20 for HDS-R wad defined as dementia. Statistical analysis was performed using Student’s t test. *p < 0.05, **p < 0.01.

Discussion

Herein, we reported that the plasma levels of AAs are altered in patients with CKD. Also, intra-brain levels of L-Ser are inversely correlated with kidney function. Moreover, the patients both with lower levels of intra-brain L-Ser and lower kidney function tend to be associated with lower JART score than the other groups. In hemodialyzed patients, plasma levels of L-Ser are also decreased in those with a lower JART score, regardless of DKD or non-DKD.

AAs plays a central role in brain functions. D-Ser contributes to postsynaptic signal transduction via NMDAR activation [20]. Therefore, various neural disorders, such as Alzheimer’s disease [21], schizophrenia [22], and epilepsy [23], are related to the disrupted D-Ser metabolism. Besides D-Ser, L-Ser is reportedly associated with the control of oxidative damage in the brain [24]. The alteration of D-3-phosphoglycerate dehydrogenase, which modulates the production of L-Ser, causes neurogenic disorders [25]. In fact, L-Ser has therapeutic potential in various neurogenic disorders, such as amyotrophic lateral sclerosis and hereditary sensory autonomic neuropathy type I [26]. Also, clinical trials for Alzheimer’s disease have been conducted with the use of L-Ser (ClinicalTrials.gov Identifier: NCT03062449). Based on these findings, lower level of intra-brain L-Ser may contribute to disrupted brain function, resulting in a lower score of JART. However, functional analysis of L-Ser is needed to clarify the pathophysiological role in brain function.

Moreover, both intra-brain levels of L-Ser and kidney function are correlated with the JART score in CKD patients. CKD patients with intra-brain levels of L-Ser below 600 nmol/g showed lower JART score than those with over 600 nmol/g. This finding indicated that low levels of intra-brain L-Ser might be a risk for impaired cognitive function in CKD patient. While we have reported that plasma levels of D-Ser are inversely correlated with kidney function [5], the plasma levels of L-Ser are similar between the patients with/without kidney dysfunction in this study. Nevertheless, intra-brain L-Ser levels decreased in accordance with the decreased kidney function, suggesting that the levels of L-Ser in circulation do not affect the intra-brain L-Ser levels directly.

As for the metabolism of L-Ser in the brain, multiple phosphorylated pathways regulate de novo synthesis of L-Ser in brain [27]. Moreover, serine racemase, which catalyzes D-Ser synthesis from L-Ser may modulate the intra-brain levels of L-Ser [17]. We performed immunostaining of SRR and DAO in the brain to evaluate the involvement of these enzymes. SRR and DAO were detected in glia cells, especially in astrocyte. The number of each positive cells was not correlated with kidney function. Based on our findings, the lower levels of intra-brain L-Ser may not be due to the regulation by SRR and DAO expression in patients with kidney dysfunction. However, the activity of these enzymes needs to be clarified to understand the intra-brain regulation of D-/L-Ser in CKD patient.

In addition, hemodialyzed patients with declined cognitive function also show lower levels of plasma L-Ser as compared to those with normal cognitive function. The percentage of D-Asn and Pro showed the increased plasma levels in non-DKD patient with dementia. Consistent with these findings, the altered plasma AAs pattern was reported in patients with Alzheimer’s disease [28]. Moreover, patients with essential tremor showed decreased plasma L-Ser levels [29]. These studies indicate that plasma levels of L-Ser could be a novel biomarker for the neurological disorders. Based on the result, plasma levels of L-Ser was associated with cognitive function, regardless of kidney function. Also, the percentage of D-Asn and Pro could reflect the cognitive function in HD patients without diabetes. However, more detailed studies with increased sample numbers are needed to clarify the possibility. Moreover, to understand the involvement of plasma L-Ser, D-/L-Asn and other AAs in brain function may strengthen their potential as novel biomarkers.

Considering that L-Ser may not be excreted in urine in hemodialyzed patients, kidney function does not affect the plasma levels of L-Ser in these cases, even though the kidney is an important organ for L-Ser production and absorption [30, 31]. In this notion, L-Ser is supplied from dietary foods, de novo synthesis, and recycling by protein degradation [27]. Very low levels of plasma Ser were reported in patients with disorders in the Ser synthesis pathway, which leads to nervous system deterioration [32-34]. Moreover, low-protein diet fed rat exhibited imbalanced AA in the blood and brain [13], suggesting that therapeutic diet for kidney disease may alter AA’s component. The mechanisms of regulation of L-Ser concentration in plasma need to be elucidated, especially in patients with CKD.

In CKD patients, plasma levels of D-Ser and D-Asn reportedly reflect kidney function [35, 36]. Our results also showed higher plasma levels and lower urine levels of D-Ser and D-Asn in CKD patients as compared to healthy control. These result might indicate that decreased urine excretion lead to the accumulation of D-Ser and D-Asn in circulation in CKD patients. However, plasma levels of L-AAs such as His, Ser, Gln, and Gly were similar between CKD patients and healthy control, even though urine levels of these AAs were lower in CKD patients. Moreover, while plasma levels of D-Ser and D-Asn were increased in CKD patients, only D-Asn showed the increased level in the brain in CKD patients. The metabolism of AAs in each organ remains to be explored especially in CKD patients.

The limitations to this study are the differences in age and gender between CKD patients and healthy control. Healthy control was younger than CKD patients, and all of them were male. In this notion, age and gender difference reportedly did not affect plasma level of Ser in Japanese [37]. However, age- and gender-matched control needs to be evaluated in future studies. Another issue is the relation of brain tumor and cognitive function. Because the tumors were detected in various lesions as shown in Table 1, the effect of tumors on cognitive function could not be excluded in patient with brain resection. Although it has also been suggested that IQ calculated by national adult reading test, the English version of JART, is less affected by cognitive impairment associated with brain diseases [38], more precise analysis is needed to clarify these points.

Taken together, decreased levels of brain and plasma L-Ser are associated with impaired cognitive function in patients with kidney dysfunction. Especially, plasma L-Ser levels may have a potential for novel biomarker of impaired cognitive function in hemodialyzed patient.

Acknowledgments

We thank the staff (KAGAMI Co., Ltd.) for technical support with 2D-HPLC and biological statistics and Ms R. Izaki for conducting the sample collection at Mizuho Hospital.

Statement of Ethics

This study was approved by the Ethics Committee of Kanazawa University Hospital and Mizuho Hospital (IRB approval No. 2893). The research was conducted in accordance with the Declaration of Helsinki. All participants were provided written informed consent and were informed about their right to withdraw from the study at any time.

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

This work was supported by JSPS KAKENHI (21K08224, YI) (21K07315 YN), Kanazawa University SAKIGAKE project 2020 (YI), and the Japan Agency for Medical Research and Development (TW).

Author Contributions

Yasunori Iwata, Yusuke Nakade, and Takashi Wada designed and performed the experiments. Masashi Kinoshita, Hemragul Sabit, Riho Nakajima, Kengo Furuichi, Norihiko Sakai, Shinji Kitajima, Tadashi Toyama, Taro Miyagawa, Akinori Hara, Miho Shimizu, Kouichi Sato, Megumi Oshima, Shiori Nakagawa, Yuta Yamamura, Hisayuki Ogura, Mitsutoshi Nakada, and Yoshitaka Koshino collected the samples and analyzed the clinical data. Hemragul Sabit performed immunostaining of brain. Masashi Mita and Maiko Nakane performed HPLC quantify the chiral animo acids. Yoshitaka Koshino provided advice on critical revision. Yasunori Iwata, Yusuke Nakade, and Takashi Wada wrote the manuscript. Mitsutoshi Nakada and Takashi Wada supervised the work. Yasunori Iwata and Yusuke Nakade contributed equally to this work.

Data Availability Statement

All data generated or analyzed during this study are included in this article and its online supplementary material. Further inquiries can be directed to the corresponding author.

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